Germacrene-D (FIG. 1) is a cyclic sesquiterpene hydrocarbon (C15H24; 7-iso-propyl-10-methyl-4-methylene-cyclodeca-5,10-diene) that is either constitutively present or induced in a wide range of plant species. Delta-Cadinene, gamma-muurolene and gamma-cadinene (FIG. 1) are cyclic sesquiterpene hydrocarbons that are either constitutively present or induced in a wide range of plant species.
The biosynthetic pathway for the sesquiterpenes branches off from the general terpenoid pathway, beginning with the allylic diphosphate ester farnesyl diphosphate (FDP, also called FPP) (Bohlmann, et al., Proc. Natl. Acad. Sci. U.S.A. 95, 4126-4133 (1998), Cane and Bowser, Bioorg. Med. Chem. Lett. 9, 1127-1132 (1999), Davis and Croteau, Top. Curr. Chem 209, 53-95 (2000)). Germacrene-D and delta-Cadinene can both be synthesised from FDP in a reaction that proceeds through a carbocation intermediate (FIG. 2) and are catalysed by the sesquiterpene synthases germacrene-D synthase (Guterman et al Plant Cell 14, 2325-2338 (2002)) and delta-cadinene synthase (Benedict et al Plant Phys 125, 1754-1765 (2001)) respectively. No gene has been isolated that has been demonstrated to produce significant quantities of gamma-muurolene or gamma cadinene. The pathway for sesquiterpene biosynthesis, the acetate/mevalonate pathway, is localised to the cytoplasm; in contrast to the pathways for monoterpene and diterpene biosynthesis, which occur in the chloroplast (Lange, et al., Proc. Natl. Acad. Sci. U.S.A 97, 13172-13177 (2000)). However recent labelling studies have shown that germacrene D in Solidago canadensis is formed predominantly via the methylerythritol phosphate pathway (Steliopoulos et al., Phytochemistry 60: 13-20 (2002)).
All known plant terpene synthases, however, whether monoterpene, sesquiterpene or diterpene, appear to be closely related. Similarities include the positioning of intron sequences (Trapp and Croteau, Genetics 158, 811-832 (2001)) and the presence of conserved sequences, such as an aspartate-rich DDXX(D,E) motif (Lesburg, et al., 8, 695-703 (1998)). This motif is involved in the binding of metal ions, usually Mg2+, that are necessary for catalysis.
Germacrene-D is considered to be a key intermediate in the biosynthesis of many sesquiterpenes (Yoshihara et al., Tetrahed Lett 27: 2263-2364 (1969); Bülow and König, Phytochem 55, 141-168 (2000)). Furthermore, it has been shown to increase attraction of and oviposition by the tobacco budworm moth Heliothis virescens (Mozuraitis et al Chem Senses 27, 505-509 (2002)) and is also a sex stimulant for the male American cockroach (Periplanata americana L.) (Nishino et al., J Insect Physiol 23, 415-419 (1977)). Germacrene-D, as a component of certain essential oils, has also been shown to possess antibacterial properties (Juteau et al Fitoterapia 73, 532-535 (2002)). Germacrene D is also a major volatile in leaves of Solidago species (Prosser et al. Phytochemistry 60, 691-702 (2002)) and in lemon basil (Iijima et al. Plant Physiology 134, 1-10 (2004)). In general the (R)-(+)-enantiomer is found in lower plants, whereas the (S)-(−) enantiomer is found in higher plants, although there are plants where both enanfiomers exist (Schmidt et al., Angewandte Chemie 110: 1479-1481 (1998); Schmidt et al., Chirality 11:353-362 (1999)). To date a gene for germacrene-D synthase has been isolated only from rose (Guterman et al Plant Cell 14, 2325-2338 (2002)), which appeared to produce germacrene-D only. Its enantiomeric form is unknown.
Cadinene is the first intermediate in the conversion by delta-cadinene synthase of FDP to sesquiterpene phytoalexins in cotton (Gossypium barbadense) (Benedict et al Plant Phys 125, 1754-1765 (2001); Davis and Essenberg, Phytochem 39, 553-567 (1995); Davis et al., Phytochem 41, 1047-1055 (1996)), and is the precursor of deoxyhemigossypol and hemigossypol defense sesquiterpenes. Alpha-cadinol has been shown to be a potent miticide against house dust mites (Chang et al., J. Med Entom. 38:455-458 (2001)). To date four cDNAs for delta-cadinene synthase have been isolated from Gossypium arboreum and characterisation of at least one of these has been reported (Chen et al, Arch. Biochem Biophys 324, 255-266 (1995); Meng et al, J. Nat. Prod. 62, 248-252 (1999)). Although gamma-cadinene and gamma-muurolene are present widely in plants, no genes encoding for proteins that allow production of any of these compounds in any quantity have been discovered.
The applicants have identified a polynucleotide encoding a multifunctional germacrene-D synthase which facilitates the production of germacrene-D, gamma-muurolene and delta- and gamma-cadinene in biofermentation processes. By manipulation of the process conditions, the applicant obtained germacrene-D or germacrene-D with a mix of delta-cadinene, gamma-cadinene and gamma-muurolene in different ratios. Minor amounts of delta-elemene, germacrene B, elemol and delta-cadinene may be coproduced with germacrene-D. Minor amounts of alpha-cubebene, alpha-ylangene, alpha-copaene, beta-cubebene, isoledene, epibicyclosesquiphellandrene and alpha-cadinol/beta-eudesmol may be coproduced under acidic conditions with major quantities of gamma-muurolene, delta-cadinene, gamma-cadinene and germacrene-D. The polynucleotide can also be used to co-ordinately manipulate production of germacrene-D, delta-elemene, germacrene-B, elemol and delta-cadinene in transgenic plants to alter fragrance/flavour characteristics and/or plant pathogen and/or insect interactions. The gene can also be used as a marker in marker assisted breeding to discover plant material with altered germacrene-D, and delta-cadinene, delta-elemene, elemol or germacrene-B composition.